Superscript choice system kit

Ten samples of brain tumor sections were selected for pre-analysis on oligonucleotide microarrays (4 samples; grade II [G2], and 6 samples grade III and IV [G3/G4]). The gene expression profile study was based on an oligonucleotide microarray method using HG-U133A 2.0 plates from Affymetrix (Santa Clara, CA, USA) according to the manufacturer’s recommendations. Synthesis of cDNA was performed with the use of SuperScript^® Choice System kit (Invitrogen Life Technologies, Waltham, MA, USA). Biotinylated cRNA was synthesized from the cDNA array using the BioArray HighYield RNA Transcript Labelling Kit (Enzo Life Sciences, Inc, Farmingdale, NY, USA) as the next step of analysis. After then the fragmentation of biotin-labeled cRNA was performed by the Sample Cleanup Module kit (Qiagen GmbH, Hilden, Germany) for 35 min at 94 °C. Obtained cRNA was hybridized to the HG-U133A microarray, labeled with phycoerythrin-streptavidin complex and scanned using GeneArray Scanner G2500A (Agilent Technologies, Inc., Santa Clara, CA, USA).

Evaluation of the changes in the expression profile of pyroptosis-related genes in the analyzed cells was carried out using the oligonucleotide microarray method. For this purpose, the HG-U133A 2.0 plate set (Affymetrix, Santa Clara, CA, USA) was used in accordance with the manufacturer’s recommendations.
In order to prepare the matrix for the analysis in the first step, cDNA synthesis was carried out with the SuperScript^® Choice System kit (Invitrogen Life Technologies, Waltham, MA, USA). In turn, the BioArray HighYield RNA Transcript Labeling Kit (Enzo Life Sciences, Inc, Farmingdale, NY, USA) was used to obtain biotinylated cRNA. Next, the labeled cRNA were subjected to a fragmentation step using the Sample Cleanup Module kit (Qiagen GmbH, Hilden, Germany). This process was carried out for 35 min at 94 °C. Hybridization of the cRNA to HG-U133A microarrays labeled with a phycoerythrin-streptavidin complex was the final step of the analysis. The scanning was performed using a GeneArray Scanner G2500A (Agilent Technologies, Inc., Santa Clara, CA, USA).